Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.
The damaged brain is largely incapable of functionally significant structural self-repair. This is due in part to the apparent failure of the mature brain to generate new neurons (Korr, 1980; Sturrock, 1982). However, the absence of neuronal production in the adult vertebrate forebrain appears to reflect not a lack of appropriate neuronal precursors, but rather their tonic inhibition and/or lack of post-mitotic trophic and migratory support. Converging lines of evidence now support the contention that neuronal and glial precursor cells are distributed widely throughout the ventricular subependymal of the adult vertebrate forebrain, persisting across a wide range of species groups (Goldman and Nottebohm, 1983; Reynolds and Weiss, 1992; Richards et al., 1992; Kirschenbaum et al., 1994; Kirschenbaum and Goldman, 1995a; reviewed in Goldman, 1995; Goldman, 1997; Goldman, 1998; Goldman and Luskin, 1998; and Gage et al., 1995). Most studies have found that the principal source of these precursors is the ventricular zone (Goldman and Nottebohm, 1983; Goldman, 1990; Goldman et al., 1992; Lois and Alvarez-Buylla, 1993; Morshead et al., 1994; Kirschenbaum et al., 1994; Kirschenbaum and Goldman, 1995), though competent neural precursors have been obtained from parenchymal sites as well (Richards et al., 1992; Palmer et al., 1995; Pincus et al., 1998). In general, adult progenitors respond to epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) with proliferative expansion (Reynolds and Weiss, 1992; Kilpatrick and Bartlett, 1995; Kuhn et al., 1997), may be multipotential (Vescovi et al., 1993; Goldman et al., 1996), and persist throughout life (Goldman et al., 1996). In rodents and humans, their neuronal daughter cells can be supported by brain-derived neurotrophic factor (BDNF) (Kirschenbaum and Goldman, 1995a), and become fully functional in vitro (Kirschenbaum et al., 1994, Pincus et al., 1998a, and Pincus et al. 1998b), like their avian counterparts (Goldman and Nedergaard, 1992).
A major impediment to both the analysis of the biology of adult neural precursors, and to their use in engraftment and transplantation studies, has been their relative scarcity in adult brain tissue, and their consequent low yield when harvested by enzymatic dissociation and purification techniques. As a result, attempts at either manipulating single adult-derived precursors or enriching them for therapeutic replacement have been difficult. The few reported successes at harvesting these cells from dissociates of adult brain, whether using avian (Goldman et al., 1992; 1996c), murine (Reynolds and Weiss, 1992), or human (Kirschenbaum et al., 1994) tissue, have all reported <1% cell survival. Thus, several groups have taken the approach of raising lines derived from single isolated precursors, continuously exposed to mitogens in serum-free suspension culture (Reynolds and Weiss, 1992; Morshead et al., 1994; Palmer et al., 1995). As a result, however, many of the basic studies of differentiation and growth control in the neural precursor population have been based upon small numbers of founder cells, passaged greatly over prolonged periods of time, under constant mitogenic stimulation. The phenotypic potential, transformation state and karyotype of these cells are all uncertain; after repetitive passage, it is unclear whether such precursor lines remain biologically representative of their parental precursors, or instead become transformants with perturbed growth and lineage control.
In order to devise a more efficient means of isolating native, unpassaged and untransformed progenitor cells from brain tissue, a strategy by which brain cells could be freely dissociated from brain tissue, then transduced in vitro with plasmid DNA bearing a fluorescent reporter gene under the control of neural progenitor cell-type specific promoters was developed (Wang et al., 1998). This permitted isolation of the elusive neuronal progenitor cell of the CNS, using the Tα1 tubulin promoter, a regulatory sequence expressed only in neuronal progenitor cells and young neurons.
However, Tα1 tubulin-based separations are limited in that they yield committed neuronal progenitors, and not the more multipotential neural progenitors, such as neural stem cells, of the adult brain, which can give rise to neurons, oligodendrocytes, and astrocytes. The existence of these neural stem cells has been reported in a number of studies of rodents (reviewed in Weiss et al., 1996), and precursors competent to generate both neurons and oligodendrocytes have been demonstrated in adult humans (Kirschenbaum et al., 1994; reviewed in Goldman, 1997). In rodents, these cells have been clonally expanded using repetitive passage and mitogenic stimulation, as described above. Nonetheless, native adult neural stem cells have never been separated and purified as such, in rodents or humans.
A strong need therefore exists for a new strategy for identifying, separating, isolating, and purifying native multipotential neural progenitor cells from brain tissue.